The primary focus of this proposal is to examine the physiological changes associated with tumor reoxygenation following single-dose and multi- fraction radiotherapy. The possibility that tumor hypoxia, or reduced oxygen content, may serve to limit the response to fractionated radiotherapy has been the subject of much interest and debate. However, the underlying mechanisms remain poorly understood. In light of the current interest in altering the basic fractionation schemes and in combining oxygen manipulative agents with fractionated therapy, it is critical that these mechanisms be clarified. The current application will examine temporal changes in tumor oxygenation and hypoxic development as a function of radiation dose and time after irradiation. Data obtained using several sophisticated techniques will be combined and analyzed in order to interpret the physiological mechanisms involved. First, tumor oxygen availability will be spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumor sections. Second, hypoxic development in relation to the tumor vasculature will be quantified in adjacent sections using immunohistochemical detection of a recently developed nitroheterocyclic hypoxia (ER5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent and immunohistological stains will be used to define the anatomical vascular densities, the fraction of the vessels containing blood flow, and the percent necrosis. Fourth, magnetic resonance spin-echo imaging of gadolinium-DTPA uptake will be incorporated to study changes in overall tumor blood perfusion. Finally, tumor radiosensitivity will be evaluated using paired survival assays to estimate the radiobiological hypoxic fraction (HF) of the tumors. Although extensive effort has been devoted to measuring the time course of changes in the tumor HF following irradiation, little is known about corresponding changes in micro-regional tumor oxygenation. In view of the complex temporal relationships among tumor vascular configuration, hemodynamic changes, radiation dose, and hypoxic development, it is critical to perform all of the measurements using carefully controlled tumor models, rather than by combining data from a variety of tumor lines, volumes, and implantation sites. In view of reported differences in vascular development, hemodynamic response, and radiosensitivity among murine, xenograft, and spontaneous tumors, a range of tumor models will be included: KHT and RIF-1 murine sarcomas, MLS and OW-1 human ovarian xenografts, and C3H spontaneous murine mammary carcinomas. Once the temporal changes in micro-regional physiology have been defined following low and high single-dose irradiation, tumors will next be analyzed during a course of five daily 2 Gy fractions. A final goal will be to combine oxygen manipulative agents with fractionated radiotherapy, in order to better understand the combined physiological effects and to provide a basis for selecting optimal time points.